Thrombokinase



May 23, N. VOLLE THRQMBOKINASE Filed July 27, 1940 2 Sheets-Sheet 1 IN VEN TOR.

ATTOENE Y y 23, 1944- N. H. VOLLE 2,349,316

THROMBOKINASE Filed July 27, 1940 2 Sheets-Sheet 2 gzigfi.

IN VEN TOR.

Patented May 23, 1944 TOMIBOKINASE Norbert H. Voll, Cincinnati, Ohio, assignmto The Kroger Food Foundation, Cincinnati, Ohio Application July 27, 1940, Serial No. 348,061

11 Claims.

This invention relates to the art of blood analysis. More particularly it pertains to the determination or diagnosis of hemorrhagic diathesis by the analytical determination of the prothrombin content of a blood sample. In its primary adaptation, the invention is directed to a novel thrombokinase product of preeminent value as a diagnostic reagent in the prothrombin analysis of blood, embracing the process and technique as well as the improved means devised for its production.

In accordance with the realization of the important status of blood mechanism in modern therapy, considerable study has been devoted to the causative factor of hemorrhagic conditions and the corollary thereof, blood coagulation.

Among the comparatively recent contributions to this art have been the development of several analytical procedures for the determination of the prothrombin content of blood; these include the techniques of Quick as well as Smith-Warner- Brinkhous, the Kato whole blood method, and the Smith bedside method. In logical sequence, the analytical possibility of prothrombin determination in blood enabled a clinical and laboratory study as to its significance and the bleeding characteristics.

The incidence of hemorrhage in jaundiced patients has ofiered a serious medical problem, especially during the post-operative state. It has now been ascertained that the immediate cause thereof in many instances is attributable to a lowering of blood prothrombin. Especial significance is attributable to this conclusion in view of the observation that a jaundiced patient before operation may reveal no tendency toward bleeding, judging from clinical or laboratory tests, but suffers serious hemorrhage subsequent to operation. This may be explained on the basis that human blood manifests a substantial factor of safety; the coagulation time, in accordance with known methods of testing, may indicate a normal value until the prothrombin content of the blood has been decreased more than 80%. The hemorrhage zone will. however, be encountered by the patient during surgery as a result of any additional liver injury due to anesthesia and/or by loss of blood. Thus, the prothrombin analysis of blood preand post-operatively suggests itself as a. basis for treating jaundiced patients to avoid such cases of hemorrhage. In addition to obstructive jaundice, hepatic injury, ulcerated colitis, and new births are a vital matter of concern with respect to prothrombin deficiency. Irrespective of the'cause of prothrombin diminution, the ultimate significance of its test resides in the fact that efiectlve treatment is available. A restoration of the prothrombin content in the blood is substantially attainable by the administration of vitamin K and bile salt; where synthetic vitamin K, which is water soluble, is employed, the use of bile salt may be dispensed with.

An essential ingredient in the above specified prothrombin analytical procedures is the subtance thromboplastin. By way of example, Quick procedure tests an oxalated plasma mixed with an excess of thromboplastin and an optimum quantity of calcium; the clotting test is a direct measure of the prothrombin content of the plasma. This technique necessitates maintaining thromboplastin and calcium constant in the blood, thereby rendering the rate of coagulation a function of the prothrombin concentration.

Various animal organs have been utilized as a source of thromboplastin; principally among these has been the animal brain, illustrated by that of the steer, dog, cat as well as the rabbit. It appears to be conceded that the precise nature of the product will vary from a given source as well as from a diversity of sources; moreover slight details in manipulation or extraction as well as chemical treatment materially affects the ultimate product characteristics. It has been found that regardless of the source of production or type of thromboplastin obtained, it has been devoid of uniformity and standardization for any given technique and has manifested general instability; thus thromboplastin resulting from acetone extraction in accordance with prior art procedures has retained its activity for a week in some cases, and suggestions have been advanced for preserving the substance for a slightly longer interval by utilizing an evacuated container.

It is accordingly apparent that the utilization of thromboplastin of the character heretofore available has resulted in a definite uncertainty as to test results involving the use of this reagent in various blood testing techniques for prothrombin content. To attain any convincing indication, it has as a matter of fact been found essential to render a plurality of tests involving differam known blood specimens for the purpose of ascertaining the activity of the thromboplastin reagent during the investigation interval.

It is an object of the invention to obviate such di-fliculties and uncertainties as hereinabove referred to by an improved method and means for producing a novel product.

An important object is to provide a new diagnostic reagent which is particularly ellective in the prothrombin determination of blood.

An additional object is to produce an improved thrombokinase product. I

A further object of the invention is to devise a novel method for producing such a product of innovation.

Still another object is to present a technique for the obtention of the animal brain which is particularly effective in the improved method and attendant novel product.

Other objects and the nature and advantages of the invention will be apparent from the following description taken in conjunction with the accompanying drawings, wherein:

Fig. 1 presents a front view in elevation of a I rabbit head, showing the severance of the skin along the median line of the head;

Fig. 2 is a similar front elevation to that shown in Fig. 1, indicating the scalp in open position, determined by the applied incisions, showing the brain within its cavity;

Fig. 3 is an elevation of the brain per se, it being shown somewhat larger than actual size;

Fig. 4 presents in vertical section a mortar containing a plurality of brains, as well as a diagrammatic indication of a pestle positioned therein;

Fig. 5 diagrammatically indicates means including a specialized suction well for separating fluid extracts from the brain mass;

Fig. 6 reveals in vertical section the structural details of the said suction well;

Fig. 7 shows a desirable colored bottle with cap attached adapted for retaining thrombokinase; and

Fig. 8 offers an elevational showing of a suitable ampoule containing a measured quantity of thrombokinase for testing a blood sample.

Referring more particularly to the details of the invention, a significant basis thereof resides in the discovery that animal brains, and more especially those of rabbits, lend themselves to a critical processing which enables the preparation of a distinctive type of thrombokinase. The procedure is predicated upon what might be termed a minimal-maximal" extraction treatment designed to:

(a) Remove the water content and ineffective lipoid ingredients asoompletely as possible with a minimal amount offextraction;

(b) Retain an optimum maximal content of effective ingredients including the lipins of antioxidant. value present in the brain mass.

For optimum results, a specialized preparatory technique has been found desirable, and embraces details for removing the brain from its skull cavity as well as manipulative features for stripping it of meninges, blood vessels, and blood clots.

It is of material importance that the rabbit brain be retained essentially free from injury by virtue of the killing of the animal, which is not the invariable result of the usual slaughtering practice. To avoid crushing of the skull and injury to the brain structure, the animal is hit sumciently hard to result in stunning, but the blows are not of such force as to damage the skull and brain. The animal is then decapitated. As an alternative procedure, under some conditions, the animal's head may be simply decapitated without the preliminary stunning step.

Referring to Figs. 1-3 of the drawings in which the rabbit head is designated by numeral 4, the

fur and skin 2 are cut along the median line 3 75 2,849,818 of the skull 4 to a point, beyond the eyes and approaching the nose, referred to as B. The relatlve position of the eyes and nose are shown as 6 and 1 respectively. Upon P eling back the fur 5 and skin 2, thereby exposing skull 4 from the point of severance I at position 9 slightly beyond the eyes, the essential incisions are made. Utilizing a bone scissors, the skull is cut along the median line 3 from the point of severance 8 to a 10 point l0 which is approximately midway between the eyes. A horizontal incision ll is then applied substantially bisecting the median line incision previously referred to. By applying vigorous force to ears l2 in opposing directions, the skull is easily opened; upon inverting or turning down the head, the brain material is readily and cleanly removedfrom its skull cavity. It should be noted that the brain H, the outlines of which are defined by dotted lines, is drawn generally to actual size in Figs. 1 and 2; the indication of Fig. 3 is, however, somewhat enlarged. By proceeding in accordance with this technique, the bulbous brain mass is obtainable in its complete normal state, essentially free from blood clots, bruises and cuts.

Subsequent to the removal of the brain from its natural locale, it is essential to subject it to a so-called stripping treatment. In accordance with the prior art practice, resort was taken to the somewhat tedious utilization of sharp tweezers for removing the undesirable substances; the time necessitated for this manner of operation varied between 5 and 15 minutes. On the other hand, by the revised technique of the present in- 5 vention, completely satisfactory results are attainable in approximately 2 minutes or less. This facilitated method utilizes the index finger and thumb of the right hand as the instruments for removing the meninges and pie. mater, etc. The 40 brain is preferably held in the palm of the left hand, and wetting by water the index finger and thumb aforementioned, they are passed over the brain surface to collect the meninges, blood clots, blood vessels, etc. After each passage of the thumb and index finger over the brain, they are immersed in a bath of cold water which immediately releases into the water the adhering sub stances. Notwithstanding the simplicity of this system of treatment, it is very critical and pernaifls stripping the entire brain, free and clean of Following the above described preparatory treatment of the brain, a plurality of brain masses is broken down to a iell form in a mortar I5 by means of a pestle I6 as shown in Fig. 4. Subsequent to extraction by a predetermined reagent, preferably acetone, with the aid of an air pressure stirrer (not shown), the liquid ex tract is separated from residual matter by the means shown in Fig. 5. This apparatus comprises essentially a ring stand support I I having slidable arm l8 which is attached to and sustains the suction well generally indicated by the numeral l9. In detail, this suction well is desirably of a material which will be uneiiected by the extracting reagent utilized. While Monel metal has proven quite satisfactory where acetone is utilized, it is not intended to be restricted thereto. Thus while the tubular screen portion 20 of the well has functioned satisfactorily when fabricated from Monel metal itmay appropriately be made from a suitably foraminated ceramic material. In the fabrication of the screen tube shown in ering meninges, blood vessels or blood clots.

Fi 6, it is essential that the solder 2| closing the longitudinal seam of the tube be similarly' unefiected by the extracting reagent. While the bottom 22 of the suction well I9 is closed, the device is open at the top, fitted with a cork 23. Through this cork stopper 23, a glass tube is snugly fitted for insertion internally of the foraminated body of the well and extends toward the bottom thereof. A rubber tube 25 issuperimposed at one end over the upper extremity of glass tube 24 while the other end is connected with a water jet ejector 29. While there is no intent to be restricted to particular dimensions or details of structure, an efiective tubular screen suction well is about 8 inches long and approxiimatelynllh inches in diameter and it is soldered along its length on side'and bottom. The glass tube it may appropriately be between 8 and 10 mm. in diameter. By the device shown, it will be apparent that the extract is withdrawn through glass tube 24 and rubber tube 25, whereas the residual material remains within the container and largely adhered to the longitudinal or peripheral surface of the suction well.

Of primary importance in the extraction of the brain mass has been discovered to be the utilization of an optimum quantity of reagent. As a result of extensive investigation, it has been ascertained that the conditions of extraction should be such as to yield a product which corresponds with l3%-l5% of the fresh brain weight. Differently stated, the results of experimental determinations are to the elfect that yields in excess of indicate under-extraction, while yields below 13% suggest that the brain material has been subjected to excessive solvent action. It has been ascertained that excesses in either direction beyond the specified range materially aiiect the characteristics of the thrombokinase produced and result in a preparation having an activity which is substantially below normal. The optimum details of extraction are in accordance with the following.

It has been determined that the weight of a rabbit brain will vary between 7% and 10 grams. The optimum standard unit volume of acetone for a single extraction treatment has been ascertained by experimentation to be approximate- 1y 25 cc. per rabbit brain. Equivalently stated, for a weight of 7% to 10 grams of brain, approximately 25 cc. of acetone represents an optimal of extracting volume; while this value may be subject to some variation, dependent upon the brain weight utilized, under no circumstances should the volume of extracting agent, such as acetone, be less than 25 cc. for a 10 gram weight of fresh brain.

To facilitate expedient extraction, a retaining vessel should be of such volume as to afford an intimate contact between the liquid and brain mass. On a production basis involving 100 rabbit brains in a given operation, it will be apparent that approximately 2500 cc. of actone will be desirable for a given extraction. A convenient receptacle for treating this quantity of material is a mortar afiording a volume content of 3 liters, or preferably somewhat larger.

In the performance of the process, the predetermined number of brains treated in accordance with the preparatory requirements, are first placed in the mortar and broken down into a jell state with the aid of a pestle. The calculated unit quantity of acetone, either C. P. or analytical reagent in quality, is then added to the mortar and the entire mass subjected to agitation "removal of acetone should desirably beincom during an interval of 15 minutes with the aid of an air pressure stirrer. At the end of the specified extraction period, the acetone is removed, preferable by utilization of suction well N in accordance with the showing of Fig. 5. The brain tissue mass is further broken down with the aid of the pestle, the calculated amount of acetone (approximately 2500 cc. per rabbit brains) is added and the agitation repeated. After a second extraction period of 15 minutes, the acetone is removed in a manner similar to that suggested in the first extraction treatment. The remaining mass is subjected to further disaggregation by the use of pestle, and the same details of extraction procedure are-repeated for a third time, with the exception that the final plete to facilitate further handling. The resultant mass containing suficient acetone to retain the fluid strength, is subjected to suction filtration through a Buchner funnel. By permitting air to pass through the funnel after the liquid hasbeen withdrawn, traces of adhering acetone are removed and the materials flaked up. The flaked material is then loosely placed into suitable receptacles exemplified by Petri dishes, and is dried at room temperature under vacuum for approximately 2 hours.

The thromkobinase product thereby attained is light and heat labile. As a protective expedient, it may accordingly be retained in a colored glass bottle 26 of green or brown coloration having a Bakelite screw cap 21. Thus enclosed the thrombokinase may be preserved under conditions of refrigeration. This desirability for refrigeration is significant for storage of the thrombokin-ase over comparatively long periods, but does not preclude its retention for reasonably short tim intervals without any substantial detriment to the activity of the product. For this purpose, it is conveniently sealed in ampoules 28, Fig. 8, for distribution of predetermined test quantities. A primary attribute of the product of this invention is, under ordinary conditions, its retention of potency as well as activity as a reagent in the various available techniques for prothrombin determination. Thrombokinase prepared as described has invariably been found to retain its effectiveness as a reagent in prothrombin diagnosis for periods of from 6 months to a year and even longer periods.

The thromboplastin substances produced in accordance with the prior art have been generally unreliable as a result of the lack of stability and uniformity as well as uncertain potency which they manifested. As above indicated, they varied widely in terms of these characteristics in view of the natural diiferences occurring in any given source of brain substance as well as inevitable differentiations in manipulation. Considering this in terms of the Quick technique, where a blood sample gave a blood clotting time of 20 seconds, there is no certainty as to whether or not this variation from the normal time of 12-13 seconds was due to the patient or due to the defectiveness of the thromboplastin. Only by means of a plurality of tests checking the material against various known specimens could it be ascertained whether or not the time of 20 seconds indicated a true condition of the patient or represented a lack of potency on the part of the thromboplastin material. Contrary to this situation, it has been found that the thrombokinase of the present invention is unequivocally reliable for prothrombin determination in accordance with the Quick technique; its uniformity and standardization characteristics are such as to render the blood clotting time ascertained acceptable as correct without the necessity of a plurality of simultaneous comparative tests.

Relative to the potency of the product, this may be attributable to the preservative characteristics of the antioxidant lipins retained in the thrombokinase by virtue of the optimal extraction procedure above described. The explanation has been advanced that the lack of stability of the prior art thromboplastin is due to a gradual deterioration of the active ingredients thereof under all essential conditions of handling and treating. When this deterioration has exceeded the point wherein the active ingredient is no longer present in an excess of that essential for a given test technique, then a satisfactory result is unattainable. Reasoning from this basis, the thrombokinase product of the present invention presumably contains such an excess of active or effective ingredients as to enable preservation for as much as a year or longer before the active ininsufficiency, or conversely, this novel thrombokinase may, as a result of the features of treatment herein disclosed, be comparatively free from such ingredients as have a tendency to promote deterioration of the active ingredient content. In this condition, the opinion has been advanced by some authoritative sources that the facilitating influence in thromboplastin is the phosphated kephalin, In any event, without intending to be restricted to any theoretical explanation of the potency of these substances, been determined as a result of experimental study of the newly prepared thrombokinase, that its strength and potency is very materially distinguishable from the prior art analogous substances. A special illustration of this is available by the indication that in lieu of the conventional 0.3 gram of thromboplastin material utilized as a basis for preparing thromboplastin extract in accordance with the Quick method of prothrombin test, a 0.1 proportion thereof or 30 milligrams of freshly prepared thrombokinasethat is, approximately 2 months old, has been utilized to give satisfactory test results. The alternative conclusions from this result are that the product of this invention contains a vastly increased proportion of active ingredient as compared with the prior art thromboplastin, or that the potency of the active ingredient in this new substance is several fold greater than that heretofore attainable in thromboplastin.

Relative to the physical characteristics of the thrombokinase as prepared by the novel procedures specified hereinabove, it is obtained as a powder of slightly pink cast. A very pungent organic odor is manifested which'is slightly suggestive of acetone.

The utilization of the described thrombokinase product will be presented in connection with the Quick procedure. To prepare thrombokinase extract, 0.3 gram of thrombokinase of the present invention is mixed with 5 cc. of physiological solution of sodium chloride containing 0.1 cc. of sodium oxalate. The mixture is incubated at 45 degrees C. for 10 minutes, then centrifuged at slow speed for 10 minutes, subsequently to which the extract is withdrawn; it is this thrombokinase extract, essentially a milky white dispersion or emulsion, which represents the diagnostic reagent actually utilized in the testing of a blood sample.

An alternative for the centrifuge separation it has conclusively 'gredlent content has deteriorated to the point of milligrams of the thromboklnase produce of the of the oxalate solution.

of the thrombokinase extract, which in view of its facility of performance may be a preferred embodiment, is the so characterized cotton plug procedure. Subsequent to the complete reagent treatment of the thrombokinase, including the incubation, a cotton wad or plug is pressed downwardly, preferably by a pipette, in the test tube retaining the mixture to separate the more solid particles of brain material from the fluid; the latter, a substantially milky white extract, may be decanted or drawn off through the pipette.

In performing the blood test, the blood sampie is freely withdrawn from a large vein by means of any known clinical instrument, or simply by utilizing a large needle and syringe. To prevent any breakdown of platelets and release of blood thromboplastin, the means resorted to for blood withdrawal should be oiled. A volume of 4.5 cc. of this venous blood is added to 0.5 cc. These substances are mixed and centrifuged to obtain clearplasma. 0.1 cc. of this plasma is added to 0.1 cc. of the thrombokinase extract in a small test tube and 0.1 cc. of calcium chloride solution is quickly admixed therewith. Retaining the test tube in a bath at 37.5 degrees C., the time lapse before the clot formation is carefully measured with a stop watch and is indicative of the prothrombin content of the blood. 7

The precise concentrations of the reagents utilized in the test as set forth by Quick are as follows: in the preparation of sodium oxalate solution, 1.34 gram of pure anhydrous sodium oxalate is dissolved in 100 cc. of distilled water; the calcium chloride utilized results from the solution of 1.11 gram of chemically pure anhydrous calcium chloride in 400 cc. of distilled water.

While this procedure described follows a strict observation of the Quick technique, as previously indicated hereinabove, it has been found that satisfactory tests are attainable by utilizing 30 present invention which has been comparatively freshly prepared in lieu of the 0.3 gram specified by Quick.

While the thrombokinase present invention has been in view of its value as a test reagent in the prothrombin diagnosis of blood, it should be understood that it manifests excellent therapeutic properties as a blood coagulant.

From the foregong, it will be apparent that the invention provides a novel procedure and excellent means for preparing a distinct improvement in thrombokinase product which is adaptable to any blood testing technique involving the conversion of prothrombin to thrombin in the presence of a thromboplastin reagent.

It will be obvious to those skilled in the art that various changes may be made without departing from the spirit of the invention and therefore the invention is not limited to what is shown in the drawings and described in the specification but only as indicated in the appended claims.

What is claimed is:

1. In the. method of producing thrombokinase, the steps which comprise disaggregating substantially complete and uninjured rabbit brains freed from meninges, blood clots and blood vessels, extracting the resulting mass with an amount of acetone corresponding to approximately 25 cc. for

type product of the described primarily each 7 /2 to 10 grams of brain, separating the fluid containing the extracting agent, twice repeating the disaggregating procedure followed by extraction with the same quantities of acetone,

and dehydrating the residue under reduced pressure and atmospheric temperature.

2. In the method of producing thrombokinase, the steps which comprise disaggregating rabbit brains freed from meninges, blood clots and blood vessels, treating the resultant mass with an amount of acetone corresponding with approximately 25 cc. for each 7 /2 to grams of brain and agitating the mixture for an interval of approximately minutes, separating the acetone fluid from the residual mass by suction, twice repeating the entire acetone extracting and separating procedure with only a partial separation of acetone fluid the last time, filtering the remaining substance and drying the residue atnormal temperature and reduced pressure.

3. In the method of producing a substance adapted as a reagent in the testing of blood, the steps which comprise disaggregating substantially complete uninjured rabbit brains freed from meninges, blood clots and blood vessels, treating the resultant mass with acetone in quantity to substantially dehydrate the mass and dissolve ineffective reagent ingredients without substantially altering the content of antioxidant and effective reagent ingredients, the resultant product being on the order of between 13 and 15% of the fresh brain weight.

4. The method of preparing thrombokinase which comprises disaggregating substantially uninjured rabbit brains freed from meninges, blood clots and blood vessels, and extracting the resultant mass with a quantity of acetone to produce a product which when dried is substantially between 13 and 15% of the fresh brain weight.

5. The method of preparing thrombokinase from substantially complete and uninJured rabbit brains which comprises passing the wetted index finger and thumb of the hand over the brain surface to collect meninges, blood clots and blood vessels, separating these substances by immersing the said finger and'thumb in cold water. disaggregating the brain, extracting the resultant mass witha quantity of acetone to produce a product which when dried is .substantially between 13% and 15%. of the fresh brain weight.

6. The method of preparing thrombokinase from substantially complete and uninjured rabbit brains which comprises passing the wetted finger of the hand over the brain surface to collect meninges, blood clots and blood vessels, separating these substances by immersing the said finger in cold water, disaggregating the brain, extracting the resultant mass with acetone between 13% and 15% of the fresh brain weight.

7. The product resulting from the process comprising disaggregating substantially complete and uninjured rabbit brains freed from meninges, blood clots and blood vessels, extracting the resulting mass with an amount of acetone corresponding to approximately 25 cc. for each 7 /2 to 10 grams of brain, separating the fluid contain-' ing the extracting agent, twice repeating the disaggregating procedure followed by extraction with the same quantities of acetone, and dehydrating the residue under reduced pressure and atmospheric temperature.

8. A therapeutic product, thrombokinase, the residue obtained by the extracting and dehydrating of rabbit brain with acetone and comprising substantially 13% to 15% of the fresh brain weight.

9. A thrombokinase extract prepared by mixing thrombokinase with physiological solution of sodium chloride containing sodium oxalate, incubating the mixture. and separating the resultant extract, the said thrombokinase being derived by the extraction of rabbit brain with acetone and comprising substantially 13% to 15% of the fresh brain weight.

10. The method of preparing thrombokinase extract which comprises disaggregating substantially complete and uninJured rabbit brains freed from meninges, blood clots and blood vessels, extracting the resultant mass with a quantity of acetone to produce a product which when substantially dried is between approximately 13% and 15% of the fresh brain weight, mixing said dried product with a physiological solution containing sodium chloride, incubating the mixture, and separating the resultant extract.

11. The method of preparing thrombokinase extract which comprises disaggregating substantially uninjured rabbit brains freed from meninges, blood clots and blood vessels, extracting the resultant mass with a quantity of acetone to produce a product which in the substantially dry state is between 13% and 15% of the fresh brain weight, mixing said dry product with a physiological solution for extracting the thrombokinase content, incubating the mixture and separating the resultant extract.

NORBERT H. vomit. 

